BACKGROUND/OBJECTIVES: Obesity impairs intestinal glucose uptake (GU) (intestinal uptake of circulating glucose from blood) and alters gut microbiome. Exercise improves intestinal insulin-stimulated GU and alters microbiome. Genetics influence the risk of obesity and gut microbiome. However, the role of genetics on the effects of exercise on intestinal GU and microbiome is unclear. METHODS: Twelve monozygotic twin pairs discordant for BMI (age 40.4 ± 4.5 years, BMI heavier 36.7 ± 6.0, leaner 29.1 ± 5.7, 8 female pairs) performed a six-month-long training intervention. Small intestine and colonic insulin-stimulated GU was studied using [F]FDG-PET and microbiota from fecal samples with 16s rRNA. RESULTS: Ten pairs completed the intervention. At baseline, heavier twins had lower small intestine and colonic GU ( < 0.05). Response to exercise differed between twins ( = 0.05), with leaner twins increasing colonic GU. Alpha and beta diversity did not differ at baseline. During the intervention, beta diversity changed significantly, most prominently at the mid-point ( < 0.01). Beta diversity changes were only significant in the leaner twins when the twin groups were analyzed separately. Exercise was associated with changes at the phylum level, mainly at the mid-point (pFDR < 0.05); at the genus level, several microbes increased, such as and (pFDR < 0.05). In type 1 analyses, many genera changes were associated with exercise, and fewer, such as , were also associated with dietary sugar consumption ( < 0.05). CONCLUSIONS: Obesity impairs insulin-stimulated intestinal GU independent of genetics. Though both twin groups exhibited some microbiota changes, most changes in insulin-stimulated colon GU and microbiota were significant in the leaner twins.